ABSTRACT
Duchenne Muscular Dystrophy (DMD) is a lethal muscle disorder, caused by mutations in the DMD gene and affects approximately 1:5000-6000 male births. In this report, we identified dysregulation of members of the Dlk1-Dio3 miRNA cluster in muscle biopsies of the GRMD dog model. Of these, we selected miR-379 for a detailed investigation because its expression is high in the muscle, and is known to be responsive to glucocorticoid, a class of anti-inflammatory drugs commonly used in DMD patients. Bioinformatics analysis predicts that miR-379 targets EIF4G2, a translational factor, which is involved in the control of mitochondrial metabolic maturation. We confirmed in myoblasts that EIF4G2 is a direct target of miR-379, and identified the DAPIT mitochondrial protein as a translational target of EIF4G2. Knocking down DAPIT in skeletal myotubes resulted in reduced ATP synthesis and myogenic differentiation. We also demonstrated that this pathway is GC-responsive since treating mice with dexamethasone resulted in reduced muscle expression of miR-379 and increased expression of EIF4G2 and DAPIT. Furthermore, miR-379 seric level, which is also elevated in the plasma of DMD patients in comparison with age-matched controls, is reduced by GC treatment. Thus, this newly identified pathway may link GC treatment to a mitochondrial response in DMD.
Subject(s)
Glucocorticoids/therapeutic use , MicroRNAs/metabolism , Mitochondria/metabolism , Muscular Dystrophy, Duchenne/drug therapy , Adenosine Triphosphate/metabolism , Animals , Binding Sites , Dexamethasone/pharmacology , Disease Models, Animal , Dogs , Eukaryotic Initiation Factor-4G/chemistry , Eukaryotic Initiation Factor-4G/genetics , Eukaryotic Initiation Factor-4G/metabolism , Gene Expression Regulation/drug effects , Humans , Mice , MicroRNAs/chemistry , Mitochondrial Proton-Translocating ATPases/antagonists & inhibitors , Mitochondrial Proton-Translocating ATPases/genetics , Mitochondrial Proton-Translocating ATPases/metabolism , Muscle, Skeletal/metabolism , Muscular Dystrophy, Duchenne/genetics , Myoblasts, Skeletal/metabolism , RNA Interference , RNA, Small Interfering/metabolismABSTRACT
During embryogenesis, the intrauterine milieu affects cell proliferation, differentiation, and function by modifying gene expression in susceptible cells, such as the pancreatic ß-cells. In this limited energy environment, mitochondrial dysfunction can lead to overproduction of reactive oxygen species (ROS) and to a decline in ß-cell function. In opposition to this toxicity, ROS are also required for insulin secretion. Here we investigated the role of ROS in ß-cell development. Surprisingly, decreasing ROS production in vivo reduced ß-cell differentiation. Moreover, in cultures of pancreatic explants, progenitors were highly sensitive to ROS stimulation and responded by generating ß-cells. ROS enhanced ß-cell differentiation through modulation of ERK1/2 signaling. Gene transfer and pharmacological manipulations, which diminish cellular ROS levels, also interfered with normal ß-cell differentiation. This study highlights the role of the redox balance on ß-cell development and provides information that will be useful for improving ß-cell production from embryonic stem cells, a step in cell therapy for diabetes.
Subject(s)
Antioxidants/pharmacology , Cell Differentiation/drug effects , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/enzymology , MAP Kinase Signaling System/drug effects , Oxidants/toxicity , Acetylcysteine/pharmacology , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology , Catalase/metabolism , Embryo, Mammalian/cytology , Epithelium/drug effects , Epithelium/metabolism , Female , Hydrogen Peroxide/toxicity , Insulin-Secreting Cells/drug effects , Nerve Tissue Proteins/metabolism , Phosphorylation/drug effects , Rats, WistarABSTRACT
The Trans-activator protein (Tat) of human immunodeficiency virus (HIV) is a pleiotropic protein involved in different aspects of AIDS pathogenesis. As a number of viral proteins Tat is suspected to disturb mitochondrial function. We prepared pure synthetic full-length Tat by native chemical ligation (NCL), and Tat peptides, to evaluate their direct effects on isolated mitochondria. Submicromolar doses of synthetic Tat cause a rapid dissipation of the mitochondrial transmembrane potential (ΔΨ(m)) as well as cytochrome c release in mitochondria isolated from mouse liver, heart, and brain. Accordingly, Tat decreases substrate oxidation by mitochondria isolated from these tissues, with oxygen uptake being initially restored by adding cytochrome c. The anion-channel inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) protects isolated mitochondria against Tat-induced mitochondrial membrane permeabilization (MMP), whereas ruthenium red, a ryanodine receptor blocker, does not. Pharmacologic inhibitors of the permeability transition pore, Bax/Bak inhibitors, and recombinant Bcl-2 and Bcl-XL proteins do not reduce Tat-induced MMP. We finally observed that Tat inhibits cytochrome c oxidase (COX) activity in disrupted mitochondria isolated from liver, heart, and brain of both mouse and human samples, making it the first described viral protein to be a potential COX inhibitor.
Subject(s)
Electron Transport Complex IV/antagonists & inhibitors , Mitochondria/drug effects , tat Gene Products, Human Immunodeficiency Virus/pharmacology , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology , Animals , Brain/drug effects , Brain/enzymology , Cytochromes c/metabolism , Electron Transport Complex IV/metabolism , Humans , Ion Transport , Liver/drug effects , Liver/enzymology , Membrane Potential, Mitochondrial , Mice , Mice, Inbred BALB C , Mitochondria/enzymology , Mitochondrial Membranes/drug effects , Mitochondrial Membranes/metabolism , Myocardium/enzymology , Oxidative Phosphorylation , Permeability , Proto-Oncogene Proteins c-bcl-2/metabolism , tat Gene Products, Human Immunodeficiency Virus/chemistry , tat Gene Products, Human Immunodeficiency Virus/physiologyABSTRACT
Brain protection of the newborn remains a challenging priority and represents a totally unmet medical need. Pharmacological inhibition of caspases appears as a promising strategy for neuroprotection. In a translational perspective, we have developed a pentapeptide-based group II caspase inhibitor, TRP601/ORPHA133563, which reaches the brain, and inhibits caspases activation, mitochondrial release of cytochrome c, and apoptosis in vivo. Single administration of TRP601 protects newborn rodent brain against excitotoxicity, hypoxia-ischemia, and perinatal arterial stroke with a 6-h therapeutic time window, and has no adverse effects on physiological parameters. Safety pharmacology investigations, and toxicology studies in rodent and canine neonates, suggest that TRP601 is a lead compound for further drug development to treat ischemic brain damage in human newborns.
Subject(s)
Caspase Inhibitors , Cysteine Proteinase Inhibitors/therapeutic use , Hypoxia-Ischemia, Brain/drug therapy , Ischemia/drug therapy , Neuroprotective Agents/therapeutic use , Oligopeptides/therapeutic use , Quinolines/pharmacology , Animals , Animals, Newborn , Apoptosis/drug effects , Binding Sites , Caspases/metabolism , Cysteine Proteinase Inhibitors/chemistry , Cytochromes c/metabolism , Disease Models, Animal , Hypoxia-Ischemia, Brain/pathology , Ischemia/pathology , Mice , Neuroprotective Agents/chemistry , Oligopeptides/chemistry , Oligopeptides/pharmacology , Quinolines/chemistry , RatsABSTRACT
The BH3 mimetic ABT737 induces autophagy by competitively disrupting the inhibitory interaction between the BH3 domain of Beclin 1 and the anti-apoptotic proteins Bcl-2 and Bcl-X(L), thereby stimulating the Beclin 1-dependent allosteric activation of the pro-autophagic lipid kinase VPS34. Here, we examined whether ABT737 stimulates other pro-autophagic signal-transduction pathways. ABT737 caused the activating phosphorylation of AMP-dependent kinase (AMPK) and of the AMPK substrate acetyl CoA carboxylase, the activating phosphorylation of several subunits of the inhibitor of NF-κB (IκB) kinase (IKK) and the hyperphosphorylation of the IKK substrate IκB, inhibition of the activity of mammalian target of rapamycin (mTOR) and consequent dephosphorylation of the mTOR substrate S6 kinase. In addition, ABT737 treatment dephosphorylates (and hence likewise inhibits) p53, glycogen synthase kinase-3 and Akt. All these effects were shared by ABT737 and another structurally unrelated BH3 mimetic, HA14-1. Functional experiments revealed that pharmacological or genetic inhibition of IKK, Sirtuin and the p53-depleting ubiquitin ligase MDM2 prevented ABT737-induced autophagy. These results point to unexpected and pleiotropic pro-autophagic effects of BH3 mimetics involving the modulation of multiple signalling pathways.
Subject(s)
Apoptosis Regulatory Proteins/agonists , Autophagy/drug effects , Biphenyl Compounds/pharmacology , Membrane Proteins/agonists , Nitrophenols/pharmacology , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Sulfonamides/pharmacology , Acetyl-CoA Carboxylase/metabolism , Beclin-1 , Benzopyrans/pharmacology , Cell Line, Tumor , Glycogen Synthase Kinase 3/metabolism , Humans , I-kappa B Kinase/metabolism , Nitriles/pharmacology , Oncogene Protein v-akt/metabolism , Phosphorylation , Phosphotransferases (Phosphate Group Acceptor)/metabolism , Piperazines/pharmacology , Proto-Oncogene Proteins c-mdm2/metabolism , Ribosomal Protein S6 Kinases/metabolism , Signal Transduction/drug effects , Sirtuins/metabolism , TOR Serine-Threonine Kinases/metabolism , Tumor Suppressor Protein p53/metabolismABSTRACT
Multiple cutaneous and uterine leiomyomatosis (MCUL)/hereditary leiomyomatosis and renal cell cancer (HLRCC) (OMIM 150800/OMIM 605839) is a rare hereditary disorder leading to the development of benign cutaneous and uterine smooth muscle tumours in young adults.(1,2) This disease is characterized by an increased risk of developing renal cell carcinomas.(3) It results from dominantly inherited autosomal mutations in the fumarate hydratase (FH) gene.(4) This gene encodes a Krebs cycle enzyme, present in both cytosolic and mitochondrial compartments, and probably acts as a tumour suppressor gene. We report a 22-year-old man affected by cutaneous leiomyomatosis associated with cutis verticis gyrata, disseminated collagenoma and Charcot-Marie-Tooth disease, who was harbouring the novel FH gene mutation c.821C > T, p.Ala274Val.
Subject(s)
Charcot-Marie-Tooth Disease/genetics , Fumarate Hydratase/genetics , Leiomyomatosis/genetics , Mutation , Skin Diseases/genetics , Skin Neoplasms/genetics , Carcinoma, Renal Cell/genetics , Collagen Diseases/genetics , Genetic Predisposition to Disease , Humans , Leiomyomatosis/enzymology , Leiomyomatosis/pathology , Male , Scalp Dermatoses/genetics , Sequence Analysis, DNA , Skin Neoplasms/enzymology , Skin Neoplasms/pathology , Young AdultABSTRACT
Apoptosis-inducing factor (AIF) has important supportive as well as potentially lethal roles in neurons. Under normal physiological conditions, AIF is a vital redox-active mitochondrial enzyme, whereas in pathological situations, it translocates from mitochondria to the nuclei of injured neurons and mediates apoptotic chromatin condensation and cell death. In this study, we reveal the existence of a brain-specific isoform of AIF, AIF2, whose expression increases as neuronal precursor cells differentiate. AIF2 arises from the utilization of the alternative exon 2b, yet uses the same remaining 15 exons as the ubiquitous AIF1 isoform. AIF1 and AIF2 are similarly imported to mitochondria in which they anchor to the inner membrane facing the intermembrane space. However, the mitochondrial inner membrane sorting signal encoded in the exon 2b of AIF2 is more hydrophobic than that of AIF1, indicating a stronger membrane anchorage of AIF2 than AIF1. AIF2 is more difficult to be desorbed from mitochondria than AIF1 on exposure to non-ionic detergents or basic pH. Furthermore, AIF2 dimerizes with AIF1, thereby preventing its release from mitochondria. Conversely, it is conceivable that a neuron-specific AIF isoform, AIF2, may have been 'designed' to be retained in mitochondria and to minimize its potential neurotoxic activity.
Subject(s)
Apoptosis Inducing Factor/metabolism , Brain/metabolism , Mitochondria/metabolism , Amino Acid Sequence , Animals , Apoptosis Inducing Factor/chemistry , Apoptosis Inducing Factor/genetics , Apoptosis Regulatory Proteins/chemistry , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Cell Differentiation , Cell Line, Tumor , Humans , Mice , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Molecular Sequence Data , Neurons/cytology , Neurons/metabolism , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Sequence AlignmentABSTRACT
Diabetes mellitus is occasionally observed in patients with skeletal muscle respiratory chain deficiency, suggesting that skeletal muscle mitochondrial dysfunction might play a pathogenic role in type 2 diabetes (T2D). In support of this hypothesis, decreased muscle mitochondrial activity has been reported in T2D patients and in mouse models of diabetes. However, recent work by several groups suggests that decreased muscle mitochondrial function may be a consequence rather than a cause of diabetes, since decreased mitochondrial function in mice affords protection from diabetes and obesity. We review the data on this controversial but important issue of potential links between mitochondrial dysfunction and diabetes.
Subject(s)
Diabetes Mellitus, Type 2/etiology , Mitochondria, Muscle/physiology , Animals , Diabetes Mellitus, Type 2/metabolism , Diabetes Mellitus, Type 2/physiopathology , Humans , Insulin-Secreting Cells/physiology , Mice , Mitochondria, Muscle/metabolism , Mitochondrial Diseases/complications , Models, Biological , Muscle, Skeletal/metabolism , Muscle, Skeletal/physiopathology , Signal Transduction/physiologyABSTRACT
Apoptosis-inducing factor (AIF) is a phylogenetically conserved redox-active flavoprotein that contributes to cell death and oxidative phosphorylation in Saccharomyces cerevisiae, Caenorhabditis elegans, mouse and humans. AIF has been characterized as a caspase-independent death effector that is activated by its translocation from mitochondria to the cytosol and nucleus. Here, we report the molecular characterization of AIF in Drosophila melanogaster, a species in which most cell deaths occur in a caspase-dependent manner. Interestingly, knockout of zygotic D. melanogaster AIF (DmAIF) expression using gene targeting resulted in decreased embryonic cell death and the persistence of differentiated neuronal cells at late embryonic stages. Although knockout embryos hatch, they undergo growth arrest at early larval stages, accompanied by mitochondrial respiratory dysfunction. Transgenic expression of DmAIF misdirected to the extramitochondrial compartment (DeltaN-DmAIF), but not wild-type DmAIF, triggered ectopic caspase activation and cell death. DeltaN-DmAIF-induced death was not blocked by removal of caspase activator Dark or transgenic expression of baculoviral caspase inhibitor p35, but was partially inhibited by Diap1 overexpression. Knockdown studies revealed that DeltaN-DmAIF interacts genetically with the redox protein thioredoxin-2. In conclusion, we show that Drosophila AIF is a mitochondrial effector of cell death that plays roles in developmentally regulated cell death and normal mitochondrial function.
Subject(s)
Apoptosis Inducing Factor/physiology , Apoptosis , Drosophila Proteins/physiology , Drosophila melanogaster/embryology , Amino Acid Sequence , Animals , Apoptosis Inducing Factor/chemistry , Apoptosis Inducing Factor/genetics , Central Nervous System/embryology , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Drosophila melanogaster/anatomy & histology , Drosophila melanogaster/metabolism , Energy Metabolism , Eye/anatomy & histology , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/genetics , Mitochondrial Proteins/physiology , Molecular Sequence Data , Mutation , Sequence Homology, Amino Acid , Thioredoxins/metabolismABSTRACT
Mitochondria contained in cancer cells exhibit two major alterations. First, they are often relatively resistant to the induction of mitochondrial membrane permeabilization (MMP), which is the rate-limiting step of the intrinsic pathway of apoptosis. The mechanisms of MMP resistance have come under close scrutiny because apoptosis resistance constitutes one of the essential hallmarks of cancer. Second, cancer cell mitochondria often exhibit a reduced oxidative phosphorylation, meaning that ATP is generated through the conversion of glucose to pyruvate and excess pyruvate is then eliminated as the waste product lactate. This glycolytic mode of energy production is even observed in conditions of high oxygen tension and is hence called anaerobic glycolysis. Here, we discuss the molecular mechanisms accounting for inhibition of the mitochondrial apoptosis pathway in neoplasia and discuss possible mechanistic links between MMP resistance and anaerobic glycolysis.
Subject(s)
Mitochondria/metabolism , Neoplasms/metabolism , Anaerobiosis , Animals , Apoptosis , Glycolysis , Humans , Mitochondrial Membranes/metabolism , Models, Biological , Neoplasms/pathology , Oxidative Phosphorylation , PermeabilityABSTRACT
The HIV-1 encoded apoptogenic protein Vpr induces mitochondrial membrane permeabilization (MMP) via interactions with the voltage-dependent anion channel (VDAC) and the adenine nucleotide translocator (ANT). We have designed a peptide, TEAM-VP, composed of two functional domains, one a tumor blood vessel RGD-like 'homing' motif and the other an MMP-inducing sequence derived from Vpr. When added to isolated mitochondria, TEAM-VP interacts with ANT and VDAC, reduces oxygen consumption and overcomes Bcl-2 protection to cause inner and outer MMP. TEAM-VP specifically recognizes cell-surface expressed alpha(V)beta(3) integrins, internalizes, temporarily localizes to lysosomes and progressively co-distributes with the mitochondrial compartment with no sign of lysosomal membrane permeabilization. Finally TEAM-VP reaches mitochondria of angiogenic endothelial cells to induce mitochondrial fission, dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)), cytochrome c release and apoptosis hallmarks. Hence, this chimeric peptide constitutes the first example of a virus-derived mitochondriotoxic compound as a candidate to kill selectively tumor neo-endothelia.
Subject(s)
Endothelial Cells/physiology , Gene Products, vpr/pharmacokinetics , Integrin alphaVbeta3/metabolism , Mitochondria/metabolism , Peptides/pharmacokinetics , Amino Acid Sequence , Animals , Apoptosis , Cell Survival , Dose-Response Relationship, Drug , Endothelial Cells/metabolism , Gene Products, vpr/pharmacology , Humans , Lysosomes/metabolism , Mice , Mice, Inbred BALB C , Mitochondrial Membranes/metabolism , Molecular Sequence Data , Peptides/pharmacology , PermeabilityABSTRACT
Mitochondrial respiratory chain disorders account for significant and varied presentations in paediatric practice. The true prevalence of these disorders in the paediatric population is still not well documented with predicted geographic variation. We report a retrospective analysis over a seven year period of cases presenting to a tertiary care centre and associated clinical features. The overall prevalence of mitochondrial disorders in our population is higher than expected (1/9,000 births), explained in part by multiple presentations in a consanguineous subgroup of the population (Irish travellers).
Subject(s)
Mitochondrial Diseases/epidemiology , Humans , Incidence , Ireland/epidemiology , Mitochondrial Diseases/diagnosis , Mitochondrial Diseases/genetics , PhenotypeABSTRACT
Cockayne syndrome is a multi-systemic, autosomal recessive disease characterised by postnatal growth failure and progressive multi-organ dysfunction. The main clinical features are severe dwarfism (<-2 SD), microcephaly (<-3 SD), psychomotor delay, sensorial loss (cataracts, pigmentary retinopathy, and deafness), and cutaneous photosensitivity. Here, 13 new cases of Cockayne syndrome are reported, which have been clinically diagnosed and confirmed using a biochemical transcription assay. The wide clinical variability, ranging from prenatal features to normal psychomotor development, is emphasised. When cardinal features are lacking, the diagnosis of Cockayne syndrome should be considered when presented with growth retardation, microcephaly, and one of the suggesting features such as enophthalmia, limb ataxia, abnormal auditory evoked responses, or increased ventricular size on cerebral imaging.
Subject(s)
Cockayne Syndrome/diagnosis , Abnormalities, Multiple/diagnosis , Cells, Cultured , Child , Child, Preschool , Cockayne Syndrome/psychology , Developmental Disabilities/diagnosis , Enophthalmos/diagnosis , Female , Fetal Growth Retardation/diagnosis , Growth Disorders/diagnosis , Humans , Infant , Male , Phenotype , Psychomotor PerformanceABSTRACT
The activity of deoxyguanosine kinase (DGUOK), a mitochondrial enzyme involved in the anabolism of mitochondrial (mt) deoxyribonucleotides, governs the maintenance of the mtDNA. Deleterious mutations of the DGUOK gene are thus associated with mtDNA depletion and result in combined deficiencies of mtDNA-encoded respiratory chain enzymes. With the aim to estimate the prevalence of DGUOK mutations in a cohort of 30 patients with hepatocerebral disease and combined respiratory chain deficiencies, we studied the DGUOK gene and identified previously unreported mutations in five families. Two patients and their affected sibs, born to non-consanguineous parents, were homozygous for a missense mutation (M1T, and L250S, respectively). One patient presented a homozygous 4 pb insertion (796 insTGAT) and two other patients, and their affected sibs, were compound heterozygous (E165V/L266R and E211G/L266R, respectively). These findings allowed us to propose prenatal diagnosis in two families. In conclusion, we observed a high prevalence of DGUOK mutations (17%) in patients with hepatic involvement and combined respiratory chain deficiencies with hepatic involvement.
Subject(s)
Mitochondrial Diseases/enzymology , Mitochondrial Diseases/genetics , Phosphotransferases (Alcohol Group Acceptor)/genetics , Amino Acid Sequence , Base Sequence , DNA/genetics , DNA Mutational Analysis , DNA, Mitochondrial/metabolism , Female , Humans , Infant , Liver/physiopathology , Male , Mitochondrial Diseases/physiopathology , Molecular Sequence Data , Mutation , Sequence Homology, Amino AcidABSTRACT
OBJECTIVES: We sought to determine the long-term clinical and biochemical outcome of newborns with mitochondrial cytopathies (MCs) and to identify possible prognostic factors that may modify the course of these diseases. MATERIAL AND METHODS: Fifty-seven newborns with MCs were identified in a retrospective review (1983-2002). We defined 2 different outcome categories: clinical (neurologic, hepatic, myopathic, and multiorganic) and biochemical (lactate level normalization or initially normal remaining unchanged, decreased but not normalized, and persistently high). We used 2 different statistical approaches: (1) survival studies depending on the initial symptoms and lactate and enzymatic deficiencies using the Kaplan-Meier method; and (2) the same variables compared with different survival age groups and clinical and biochemical outcome categories using the chi2 test. RESULTS: Thirty-three patients died (57.8%), 12 remain alive (21%), and 12 were lost in the follow-up; 6 of them are currently older than 4 years. Most of the patients manifested multiorganic disease (64.8%) and high lactate level (77.1%) over time. Children surviving to 2.5 to 3 years of age were more likely to survive for a long period of time. Initial neurologic and hepatic presentation increased the risk to develop neurologic disease and severe persistent hyperlactacidemia, respectively. Initial severe hyperlactacidemia and combined enzyme deficiencies were significant risk factors for higher mortality and multiorganic disorders. Two patients with exclusively myopathic outcome are alive and cognitively normal at 12 years of life. CONCLUSIONS: Children with neonatal-onset MCs have very high mortality and poor prospects. However, some with life-threatening presentations may gradually improve, giving rise to less severe diseases. Those with exclusively myopathic symptoms have a better prognosis.
Subject(s)
Infant, Newborn, Diseases , Mitochondrial Myopathies/complications , Child, Preschool , Disease Progression , Female , Follow-Up Studies , Humans , Infant , Infant, Newborn , Infant, Newborn, Diseases/blood , Infant, Newborn, Diseases/diagnosis , Infant, Newborn, Diseases/mortality , Lactic Acid/blood , Male , Mitochondrial Myopathies/blood , Mitochondrial Myopathies/diagnosis , Mitochondrial Myopathies/mortality , PrognosisABSTRACT
Mitochondrial succinate dehydrogenase (SDH) consists merely of four nuclearly encoded subunits. It participates in the electron transfer in the respiratory chain and in succinate catabolism in the Krebs cycle. Mutations in the four genes, SDHA, B, C and D, have been reported, resulting in strikingly diverse clinical presentations. So far, SDHA mutations have been reported to cause an encephalomyopathy in childhood, while mutations in the genes encoding the other three subunits have been associated only with tumour formation. Following a brief description of SDH genes and subunits, we examine the properties and roles of SDH in the mitochondria. This allows further discussion of the several hypotheses proposed to account for the different clinical presentations resulting from impaired activity of the enzyme. Finally we stress the importance of SDH as a target and/or marker in a number of diseases and the need to better delineate the consequences of SDH deficiency in humans.
Subject(s)
Succinate Dehydrogenase/deficiency , Succinic Acid/metabolism , Humans , Metabolism, Inborn Errors/genetics , Metabolism, Inborn Errors/metabolism , Mutation , Succinate Dehydrogenase/geneticsABSTRACT
The nuclear-encoded Krebs cycle enzymes, fumarate hydratase (FH) and succinate dehydrogenase (SDHB, -C and -D), act as tumour suppressors. Germline mutations in FH predispose individuals to leiomyomas and renal cell cancer (HLRCC), whereas mutations in SDH cause paragangliomas and phaeochromocytomas (HPGL). In this study, we have shown that FH-deficient cells and tumours accumulate fumarate and, to a lesser extent, succinate. SDH-deficient tumours principally accumulate succinate. In situ analyses showed that these tumours also have over-expression of hypoxia-inducible factor 1alpha (HIF1alpha), activation of HIF1alphatargets (such as vascular endothelial growth factor) and high microvessel density. We found no evidence of increased reactive oxygen species in our cells. Our data provide in vivo evidence to support the hypothesis that increased succinate and/or fumarate causes stabilization of HIF1alpha a plausible mechanism, inhibition of HIF prolyl hydroxylases, has previously been suggested by in vitro studies. The basic mechanism of tumorigenesis in HPGL and HLRCC is likely to be pseudo-hypoxic drive, just as it is in von Hippel-Lindau syndrome.
Subject(s)
Fumarate Hydratase/genetics , Germ-Line Mutation , Succinate Dehydrogenase/genetics , Succinate Dehydrogenase/metabolism , Carcinoma, Renal Cell/metabolism , Citric Acid Cycle/physiology , Female , Fumarate Hydratase/metabolism , Humans , Leiomyoma/genetics , Leiomyoma/metabolism , Neoplasms/genetics , Neoplasms/metabolism , Paraganglioma/genetics , Paraganglioma/metabolism , Tumor Cells, Cultured , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Germline mutations in the succinate dehydrogenase (SDH) (mitochondrial respiratory chain complex II) subunit B gene, SDHB, cause susceptibility to head and neck paraganglioma and phaeochromocytoma. Previously, we did not identify somatic SDHB mutations in sporadic phaeochromocytoma, but SDHB maps to 1p36, a region of frequent loss of heterozygosity (LOH) in neuroblastoma as well. Hence, to evaluate SDHB as a candidate neuroblastoma tumour suppressor gene (TSG) we performed mutation analysis in 46 primary neuroblastomas by direct sequencing, but did not identify germline or somatic SDHB mutations. As TSGs such as RASSF1A are frequently inactivated by promoter region hypermethylation, we designed a methylation-sensitive PCR-based assay to detect SDHB promoter region methylation. In 21% of primary neuroblastomas and 32% of phaeochromocytomas (32%) methylated (and unmethylated) alleles were detected. Although promoter region methylation was also detected in two neuroblastoma cell lines, this was not associated with silencing of SDHB expression, and treatment with a demethylating agent (5-azacytidine) did not increase SDH activity. These findings suggest that although germline SDHB mutations are an important cause of phaeochromocytoma susceptibility, somatic inactivation of SDHB does not have a major role in sporadic neural crest tumours and SDHB is not the target of 1p36 allele loss in neuroblastoma and phaeochromocytoma.
Subject(s)
DNA Methylation , Mutation , Neuroblastoma/genetics , Pheochromocytoma/genetics , Protein Subunits/genetics , Succinate Dehydrogenase/genetics , Base Sequence , Cell Line, Tumor , Gene Silencing , Humans , Iron-Sulfur Proteins , Loss of Heterozygosity , Molecular Sequence Data , Neural Crest , Promoter Regions, GeneticABSTRACT
An eight-week-old infant, the fourth child of consanguineous parents presented with intractable neonatal seizures. The mother had two previous miscarriages. The infant initially presented on day one with multifocal myoclonus, complex partial and generalised tonic-clonic seizures. On examination, there were dysmorphic hands and feet, with absent nails and terminal phalanges of the fingers and toes, hepatomegaly, marked axial and peripheral hypotonia and severe global developmental delay. Ophthalmological assessment showed 'salt and pepper' pigmentary retinopathy. The urinary organic acid profile revealed a marked increase in tricarboxylic acid metabolites. Urinary phosphate reabsorption was reduced at 84%. Type I fibre atrophy was seen on muscle histology, and a cytochrome c oxidase deficiency was found only on enzymology of liver tissue. Limb malformations associated with respiratory chain defects have rarely been reported. To our knowledge, this child has the most severe limb anomaly associated with a tissue-specific complex IV respiratory chain defect.
Subject(s)
Abnormalities, Multiple/genetics , Craniofacial Abnormalities/genetics , Cytochrome-c Oxidase Deficiency/genetics , Electron Transport Complex IV/genetics , Epilepsy, Benign Neonatal/genetics , Fingers/abnormalities , Liver/enzymology , Toes/abnormalities , Abnormalities, Multiple/diagnosis , Consanguinity , Craniofacial Abnormalities/diagnosis , Cytochrome-c Oxidase Deficiency/diagnosis , DNA Mutational Analysis , Developmental Disabilities/diagnosis , Developmental Disabilities/genetics , Diagnosis, Differential , Epilepsies, Myoclonic/diagnosis , Epilepsies, Myoclonic/genetics , Epilepsy, Benign Neonatal/diagnosis , Epilepsy, Complex Partial/diagnosis , Epilepsy, Complex Partial/genetics , Epilepsy, Tonic-Clonic/diagnosis , Epilepsy, Tonic-Clonic/genetics , Humans , Infant , Male , Membrane Proteins/genetics , Molecular Chaperones , Muscle Hypotonia/diagnosis , Muscle Hypotonia/genetics , Phenotype , Status Epilepticus/diagnosis , Status Epilepticus/genetics , Tricarboxylic Acids/urineABSTRACT
Respiratory chain complex I deficiency represents a genetically heterogeneous group of diseases resulting from mutations in mitochondrial or nuclear genes. Mutations have been reported in 13 of the 14 subunits encoding the core of complex I (seven mitochondrial and six nuclear genes) and these result in Leigh or Leigh-like syndromes or cardiomyopathy. In this study, a combination of denaturing high performance liquid chromatography and sequence analysis was used to study the NDUFS3 gene in a series of complex I deficient patients. Mutations found in this gene (NADH dehydrogenase iron-sulphur protein 3), coding for the seventh and last subunit of complex I core, were shown to cause late onset Leigh syndrome, optic atrophy, and complex I deficiency. A biochemical diagnosis of complex I deficiency on cultured amniocytes from a later pregnancy was confirmed through the identification of disease causing NDUFS3 mutations in these cells. While mutations in the NDUFS3 gene thus result in Leigh syndrome, a dissimilar clinical phenotype is observed in mutations in the NDUFV2 and NDUFS2 genes, resulting in encephalomyopathy and cardiomyopathy. The reasons for these differences are uncertain.